department of pharmacology, chang gung university, tao ... · and blood-brain barrier (bbb)...

*Department of Pharmacology, Chang Gung University, Tao-Yuan, Taiwan

�Department of Nursing, Chang Gung Institute of Technology, Tao-Yuan, Taiwan

Endothelial cells are known to produce vasoactive peptidesthat contribute to vasotone regulation. One of such vaso-active mediators is the endothelin (ET) family, composed ofET-1, -2, and -3 that cause vasoconstriction. Among the ETmembers, the bioactivity of ET-1 is considered as a potentvasoconstrictor and pro-inflammatory peptide and has beenimplicated in the development of cardiovascular diseases(Levin 1995; Bohm and Pernow 2007). ET-1 exerts itsbiological effects via two types of ET receptor, ET type A(ETA) and type B (ETB), which are members of G protein-coupled receptor (GPCR) superfamily (Rubanyi and Polok-off 1994). Besides the significance in cardiovascularpathology, ET-1 plays a potential role in either the normaldevelopment or in CNS diseases. Several lines of evidencesuggest the substantial role of ET-1 in brain inflammation.Hypoxic/ischemic injury of the brain may induce release ofET-1 from endothelial cells (Chen et al. 2001) and astro-cytes (Hasselblatt et al. 2001). On astrocytes, the ETB

receptor is predominantly expressed and may mediate

astrocytic hypertrophy in the injured CNS (Nakagomi et al.2000; Rogers et al. 2003). Current data have furtherdemonstrated that over-expression of ET-1 on astrocytes

Received December 24, 2009; revised manuscript received February 8,2010; accepted February 28, 2010.Address correspondence and reprint requests to Dr. Chuen-Mao Yang,

Department of Physiology and Pharmacology, College of Medicine,Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-San, Tao-Yuan,Taiwan. E-mail: [email protected] used: AP-1, activator protein-1; BBB, blood-brain bar-

rier; DMEM, Dulbecco’s modified Eagle’s medium; ECL, enhancedchemiluminescence; Elk-1, Ets-like kinase; ET, endothelin; F-12, Ham’snutrient mixture F-12; GAPDH, glyceraldehyde-3-phosphate dehy-drogenase; GFAP, glial fibrillary acidic protein; GPCR, G protein-coupledreceptor; IKK, I Kappa B kinase; IL-1b, interleukin-1b; MEK1/2, mito-gen-activated protein kinase kinase 1/2; MMP, matrix metalloproteinase;NF-jB, nuclear factor-kappa B; RBA-1, rat brain astrocyte-1; S6C, sa-rafotoxin 6C; SDS–PAGE, sodium doecyl sulfate–polyacrylamide gelelectrophoresis; shRNA, short hairpin RNA; siRNA, small interferingRNA; TIMP, tissue inhibitors of MMPs; TSIIA, tanshinone IIA.

Abstract

The bioactivity of endothelin-1 (ET-1) has been suggested in

the development of CNS diseases, including disturbance of

water homeostasis and blood-brain barrier integrity. Recent

studies suggest that hypoxic/ischemic injury of the brain in-

duces release of ET-1, behaving through a G-protein coupled

ET receptor family. The deleterious effects of ET-1 on astro-

cytes may aggravate brain inflammation. Increased plasma

levels of matrix metalloproteinases (MMPs), in particular

MMP-9, have been observed in patients with neuroinflam-

matory disorders. However, the detailed mechanisms under-

lying ET-1-induced MMP-9 expression remain unknown. In

this study, the data obtained with zymographic, western blot-

ting, real-time PCR, and immunofluorescent staining analyses

showed that ET-1-induced MMP-9 expression was mediated

through an ETB-dependent transcriptional activation.

Engagement of Gi/o- and Gq-coupled ETB receptor by ET-1 led

to activation of p42/p44 MAPK and then activated transcrip-

tion factors including Ets-like kinase, nuclear factor-kappa B,

and activator protein-1 (c-Jun/c-Fos). These activated tran-

scription factors translocated into nucleus and bound to their

corresponding binding sites in MMP-9 promoter, thereby

turning on MMP-9 gene transcription. Eventually, up-regula-

tion of MMP-9 by ET-1 enhanced the migration of astrocytes.

Taken together, these results suggested that in astrocytes,

activation of Ets-like kinase, nuclear factor-kappa B, and

activator protein-1 by ETB-dependent p42/p44 MAPK signal-

ing is necessary for ET-1-induced MMP-9 gene up-regulation.

Understanding the mechanisms of MMP-9 expression and

functional changes regulated by ET-1/ETB system on astro-

cytes may provide rational therapeutic interventions for brain

injury associated with increased MMP-9 expression.

Keywords: AP-1, astrocytes, Elk-1, endothelin-1, matrix

metalloproteinases, NF-jB, p42/p44 mitogen-activated pro-

tein kinase.

J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06680.x

JOURNAL OF NEUROCHEMISTRY | 2010 doi: 10.1111/j.1471-4159.2010.06680.x

� 2010 The AuthorsJournal Compilation � 2010 International Society for Neurochemistry, J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06680.x 1

has deleterious effects on water homeostasis, cerebral edemaand blood-brain barrier (BBB) integrity, which contribute tomore severe ischemic brain injury like stroke (Lo et al.2005). Moreover, a study model based on rabbit optic nervesuggests that ET-1 induces a hypertrophy of ETB/glialfibrillary acid protein (GFAP)-immunoreactive astrocytes, atypical characteristic of astrogliosis, in the normal opticnerve, leading to glial scar formation following CNS injury(Rogers et al. 2003). Accumulating evidence suggests thatcytokine production may be involved in the effects of ET-1in brain inflammation. It has been demonstrated thatendothelial ET-1 induces interleukin-1b (IL-1b) secretionby astrocytes, which directly contributes to BBB breakdownduring CNS inflammation (Didier et al. 2003). However, thedetailed mechanisms responsible for ET-1 action remain tobe elucidated.

The matrix metalloproteinase (MMP) family is zinc-dependent proteinases which are recognized as crucialdeterminants in normal development and wound healing, aswell as in pathophysiological implications, such as athero-sclerosis, metastasis, and inflammation. Contributions ofMMPs, MMP-9 especially, to the CNS diseases have beenidentified in the progression of neural dysfunction andneuroinflammation in the CNS (Yong et al. 1998, 2001;Aoki et al. 2002; Rosenberg 2002). The expression ofMMP-9 is induced by extracellular stimuli such ascytokines, endotoxins, and growth factors in malignantglioma and neurological diseases (Yong et al. 1998; Hark-ness et al. 2000; Rosenberg 2002). Moreover, IL-1b,bradykinin and endotoxins have been reported to up-regulate MMP-9 expression and activity in cultured ratastrocytes (Lee et al. 2003; Hsieh et al. 2004; Wu et al.2004), indicating that the expression and activation ofMMP-9 may be modulated by various factors duringneuroinflammation.

There is evidence to support the role of MMP-9 in thepathogenesis of CNS disorders (Yong et al. 2001; Rosenberg2002; Gu et al. 2005). The activity of MMP-9 is strictlyregulated at diverse levels, including transcription, transla-tion, activation, and inhibition. Moreover, the MMP-9promoter has been identified to contain potential bindingelements for recognition of transcription factors such asnuclear factor-kappa B (NF-jB), Ets, and activator protein-1(AP-1) families (Yong et al. 2001; Rosenberg 2002). How-ever, the roles of these transcription factors in astrocyticMMP-9 gene induced by ET-1 remain unclear.

In this study, we investigated the molecular mechanismsunderlying ET-1-induced MMP-9 expression in rat brainastrocytes. These findings suggested that ET-1 inducedMMP-9 expression at the transcriptional and translationallevels, which was mediated through the ETB receptor-dependent activation of p42/p44 MAPK/Ets-like kinase(Elk-1), NF-jB, and AP-1 pathways, leading to cell migra-tion in rat brain astrocytes.

Materials and methods

MaterialsDulbecco’s modified Eagle’s medium (DMEM)/Ham’s nutrient

mixture F-12 (F-12) medium, fetal bovine serum, and TRIzol were

from Invitrogen (Carlsbad, CA, USA). Hybond C membrane and

enhanced chemiluminescence (ECL) western blotting detection

system were from GE Healthcare Biosciences (Buckinghamshire,

UK). MMP-9 antibody was from NeoMarker (Fremont, CA, USA).

Phospho-(Thr202/Tyr204)-p42/p44 MAPK, and phospho-(Ser176/

180)-IKKa/b antibody kits were from Cell Signaling (Danver, MA,

USA). Phospho-(Ser383)-Elk-1, p42, I Kappa B kinase (IKK) a/b,p65, phospho-c-Jun, c-Jun, and c-Fos antibody kits were from Santa

Cruz (Santa Cruz, CA, USA). Anti-glyceraldehyde-3-phosphate

dehydrogenase (GAPDH) antibody was from Biogenesis (Boume-

mouth, UK). MMP-2/9i, BQ-123, BQ-788, GP antagonist-2, GP

antagonist-2A, U0126, Bay11-7082, and tanshinone IIA were from

Biomol (Plymouth Meeting, PA, USA). Bicinchoninic acid protein

assay reagent was from Pierce (Rockford, IL, USA). Enzymes, ET-

1, XTT assay kit, and other chemicals were from Sigma (St. Louis,

MO, USA).

Rat brain astrocyte cultureRat brain astrocyte-1 (RBA-1) cells were used throughout this study.

This cell line was originated from a primary astrocyte culture of

neonatal rat cerebrum and naturally developed through successive

cell passages (Jou et al. 1985). Staining of RBA-1 with the astrocyte-specific marker, GFAP, showed over 95% positive staining. In this

study, the RBA-1 cells were used within 40 passages that show

normal cellular morphological characteristics and had steady growth

and proliferation in the monolayer system. Cells were cultured and

treated as previously described (Hsieh et al. 2004). Primary astrocyte

cultures were prepared from the cortex of 6-day-old Wistar rat pups,

handled according to the guidelines of Animal Care Committee of

Chang Gung University and NIH Guides for the Care and Use of

Laboratory. As previously described (Hsieh et al. 2008), cortex was

dissected and dissociated by mechanical chopping and trypsinization

(0.125% trypsin at 37�C for 30 min) and then filtered through a

75 lm pore size sterilized nylon mesh to obtain a single-cell

suspension. Cells were plated into poly-L-lysine solution-coated

plastic culture dishes and routinely cultured in DMEM/F-12 medium

containing 10% fetal bovine serum at 37�C in a humidified 5% CO2

atmosphere. Forty minutes later, the unattached cells (i.e., astroglia

cells) were transferred to another plastic dish with the same medium.

The cells were plated on 12-well plates and 10-cm culture dishes for

MMP gelatin zymography and PCR, respectively. The culture

medium was changed every 3 days. The purity of primary astrocyte

cultures was assessed with the astrocyte-specific marker, GFAP,

showing over 90% GFAP-positive astrocytes.

MMP gelatin zymographyAfter ET-1 treatment, the culture medium was collected, mixed with

equal amounts of non-reduced sample buffer and electrophoresed on

10% sodium doecyl sulfate–polyacrylamide gel electrophoresis

(SDS–PAGE) containing 1 mg/mL gelatin as a protease substrate.

Following electrophoresis, gelatinolytic activity was determined as

previously described (Hsieh et al. 2004). Mixed human MMP-2 and

MMP-9 standards (Chemicon, Temecula, CA, USA) are used as

Journal Compilation � 2010 International Society for Neurochemistry, J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06680.x� 2010 The Authors

2 | H.-H. Wang et al.

positive controls. Because cleaved MMPs were not reliably

detectable, only proform zymogens were quantified. When inhib-

itors were used, they were added 1 h prior to the application of

ET-1. Treatment of RBA-1 cells with pharmacological inhibitors

alone had no significant effect on cell viability determined by an

XTT assay (data not shown).

Total RNA extraction and gene expression studiesFor RT-PCR analysis, total RNA was extracted from RBA-1 cells

stimulated by ET-1 as previously described (Hsieh et al. 2004). ThecDNA obtained from 0.5 lg total RNA was used as a template for

PCR amplification. Oligonucleotide primers were designed based on

Genbank entries for rat MMP-9, tissue inhibitors of MMP (TIMP)-1,

TIMP-2, c-fos, and b-actin. The following primers were used for

amplification reaction: for MMP-9: 5¢-AGTTTGGTGTCGCGGAGCAC-3¢ (sense), 5¢-TACATGAGCGCTTCCGGCAC-3¢ (anti-

sense); for TIMP-1: 5¢-CTGGCATCCTCTTGTTGCTA-3¢ (sense),5¢-GTAGCCCTTCTCAGAGCCCA-3¢ (anti-sense); for TIMP-2: 5¢-CGGACCCAGGCCCCTAGCGC-3¢ (sense), 5¢-AGCATGGGAT-CATAGGGCAG-3¢ (anti-sense); for c-fos: 5¢-AGACGAAGGA-AGACGTGTAAGCACTGCAGCT-3¢ (sense), 5¢-AAGGAGAATC-CGAAGGGAAAGGAATAAGATG-3¢ (anti-sense); for b-actin: 5¢-GAACCCTAAGGCCAACCGTG-3¢ (sense), 5¢-TGGCATAGAG-GTCTTTACGG-3¢ (anti-sense). PCR mixes contained 10 lL of 5·PCR buffer, 1.25 mM of each dNTP, 100 pmol of each forward and

reverse primer, and 2.5 units of Taq polymerase (Takara, Shiga,

Japan). The final reaction volume was 50 lL. Amplification was

performed in 30 cycles at 55�C, 30 s; 72�C, 1 min; 94�C, 30 s.

After the last cycle, all samples were incubated for an additional

10 min at 72�C. PCR fragments were analyzed on 2% agarose 1·Tris-acetate-EDTA (TAE) gel containing ethidium bromide and their

size was compared to a molecular weight marker. Amplification of

b-actin, a relatively invariant internal reference RNA, was per-

formed in parallel, and cDNA amounts were standardized to

equivalent b-actin mRNA levels. These primer sets specifically

recognized only the genes of interest as indicated by amplification of

a single band of the expected size (754 bp for MMP-9, 600 bp for c-

fos, and 514 bp for b-actin) and direct sequence analysis of the PCR

products. Real-time PCR was performed with the TaqMan gene

expression assay system, using primer and probe mixes for MMP-9,

ETA, ETB, and endogenous GAPDH control genes. The primers

were: for MMP-9: 5¢-TGATGCCATTGCTGATATCCA-3¢ (sense),5¢-CGGATCCTCAAAGGCTGAGT-3¢ (anti-sense); for ETA: 5¢-AATACAAGGGCGAGCAGCAC-3¢ (sense), 5¢-GCAAGCTCC-CATTCCTTCTGT-3¢ (anti-sense); for ETB: 5¢-AGACGAGAAG-TGGCCAAGACA-3¢ (sense), 5¢-GGAATTCAAAGAAGCCATGT-TG-3¢ (anti-sense); for GAPDH: 5¢-AACTTTGGCATCGTGGA-AGG-3¢ (sense), 5¢-GTGGATGCAGGGATGATGTTC-3¢ (anti-

sense). PCRs were performed using the 7500 Real-Time PCR

System (Applied Biosystems, Foster City, CA, USA). Relative gene

expression was determined by the DDCt method, where Ct meant

threshold cycle. All experiments were performed in triplicate.

ELISA analysisThe culture medium samples were collected from ET-1-incubated

RBA-1 cells and analyzed for MMP-9 levels using the rat MMP-9

ELISA kit (Cusabio Biotech, Wuhan, Hubei, China) according to

the manufacturer’s instructions.

Migration assayRat brain astrocyte-1 cell monolayers were grown on coverslips in

6-well culture plates. After starvation with serum-free DMEM/F-12

medium for 24 h, the coverslips were inverted with the cell

monolayers facing down onto a new culture plate. Serum-free

DMEM/F-12 medium with or without ET-1 was added to each dish

as indicated after pre-treatment with the inhibitors for 1 h.

Hydroxyurea, an inhibitor of DNA synthesis (Yarbro 1992), was

added to prevent cell proliferation during the period of observation.

Images of migratory cells from the coverslip boundary were

observed and acquired at 0 and 48 h with a digital camera and a

light microscope (Olympus, Tokyo, Japan). Number of migratory

cells was counted from the resulting four phase images for each

point and then averaged for each experimental condition. The data

presented are generated from three separate assays.

Western blotting analysis of MMP-9Conditioned media derived from untreated or ET-1-treated RBA-1

cells were mixed with equal amounts of reduced sample buffer at

95�C for 5 min before electrophoresis with 10% SDS–PAGE. The

resolved band was electrotransferred onto nitrocellulose membrane

which was incubated overnight at 4�C with monoclonal antibody

against MMP-9 as described by Hsieh et al. (2004).

Preparation of cell extracts and western blot analysisGrowth-arrested RBA-1 cells were incubated with ET-1 at 37�C for

various times. The cells were washed with ice-cold phosphate-

buffered saline, scraped, and collected by centrifugation at 45 000 gfor 1 h at 4�C to yield the whole cell extract, as described previously

(Hsieh et al. 2004). Samples were denatured, subjected to SDS–

PAGE using a 10% (w/v) running gel, and transferred to

nitrocellulose membrane. Membranes were incubated overnight

using anti-phospho-p42/p44 MAPK, phospho-Elk-1, phospho-

IKKa/b, or phospho-c-Jun antibody. Membranes were washed with

Tris-Tween buffered saline (TTBS) four times for 5 min each,

incubated with a 1 : 2000 dilution of anti-rabbit horseradish

peroxidase antibody for 1 h. The immunoreactive bands detected

by ECL reagents were developed by Hyperfilm-ECL.

Immunofluorescence stainingRat brain astrocyte-1 cells were plated on 6-well culture plates with

coverslips. Cells were treated with 100 nM ET-1 and washed twice

with ice-cold phosphate-buffered saline. Immunofluorescence stain-

ing using a primary anti-p65 monoclonal antibody was performed as

described previously (Hsieh et al. 2004).

Plasmid construction, transient transfection, and promoter assaysThe rat MMP-9 promoter was constructed as previously described

(Eberhardt et al. 2002) with some modifications. The upstream

region ()1280 to +108) of the rat MMP-9 promoter was cloned into

the pGL3-basic vector containing the luciferase reporter system.

pGL-MMP-9-DEts and pGL-MMP-9-DAP1 were constructed as

previously described (Wang et al. 2010; Wu et al. 2009). Introduc-tion of a double-point mutation into the NF-jB-binding site (jBdomain; GGAATTCC to GGAATTGG) to generate pGL-MMP-9-

DjB (mt-jB-MMP-9) was performed using the following (forward)

primer: 5¢-GGGTTGCCCCGTGGAATTGGCCCAAATCCTGC-3¢(corresponding to a region from )572 to )541). The underlined

� 2010 The AuthorsJournal Compilation � 2010 International Society for Neurochemistry, J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06680.x

Endothelin-1 induces matrix metalloproteinase-9 expression in astrocytes | 3

nucleotides indicate the positions of substituted bases. All plasmids

were prepared by using QIAGEN plasmid DNA preparation kits

(Valencia, CA, USA). The dominant negative mutant of IKKa/b,short hairpin RNA (shRNA) for p42, c-Jun, and c-Fos, small

interfering RNA (siRNA) for p65 (Rela-RSS358497, Rela-

RSS317831, Rela-RSS317830; Invitrogen), and MMP-9 promoter

reporter constructs were transfected into RBA-1 cells using the

LipofetamineTM RNAiMAX reagent according to the instructions of

manufacture (Invitrogen). The transfection efficiency (�60%) was

determined by transfection with enhanced green fluorescent protein

(GFP). To assess promoter activity, cells were collected and

disrupted by sonication in lysis buffer (25 mM Tris-phosphate, pH

7.8, 2 mM EDTA, 1% Triton X-100, and 10% glycerol). After

centrifugation, aliquots of the supernatants were tested for luciferase

activity using a luciferase assay system. Firefly luciferase activities

were standardized to b-galactosidase activity.

Analysis of dataConcentration-effect curves were made using GraphPad Prism

Program (GraphPad, San Diego, CA, USA). Quantitative data were

analyzed by one-way ANOVA followed by Tukey’s honestly

significant difference tests between individual groups. Data were

expressed as mean ± SEM. A value of p < 0.05 was considered

significant.

Results

ET-1-induced proMMP-9 up-regulation enhances RBA-1cell migrationTo investigate the effects of ET-1 on proMMP-9 expression,RBA-1 cells were treated with various concentration of ET-1for the indicated time intervals. The condition media werecollected and analyzed by gelatin zymography. As shown inFig. 1(a), ET-1 induced proMMP-9 expression in a concen-tration- and time-dependent manner. There was a significantincrease within 16 h and sustained over 24 h. In contrast, theexpression of proMMP-2 was not significantly changedduring incubation with ET-1 (Fig. 1a). We also determinedwhether the regulatory proteins of MMP activity such asTIMPs could be regulated by ET-1 in RBA-1 cells. The

Time (h)

0ET-1

100 nM

10 nM

1 nM

100 nM# #

##

8

Den

sito

met

ry u

nits

(fol

d of

bas

al) 6

4

2

0

MMP-9

TIMP-1

TIMP-2

0 2 4 6 16 24

125

100

75

50

Mig

rato

ry c

ells

(no.

of

cells

)

25

0Basal – MMP-2/9i

ET-1/48 h

#

ET-1, time (h)

b-actin

2 4 6 16

ET-1 incubation time (h)

24

10 nM1 nM

2 4 6 16 24

18 #

**

12

Rel

ativ

e ge

ne e

xpre

ssio

n(f

old

of b

asal

)M

MP

-9 in

cul

ture

med

ium

(pg/

mL

)

6

0

*

*

9

12

6

3

00 16 24

ET-1 time (h)

20 4

ET-1/time (h)

6 16 24

proMMP-9

proMMP-2

(a)

(b)

(c)

(d)

(e)

Fig. 1 ET-1 induced proMMP-9 expression

and astrocytic migration. (a) Time and

concentration dependence of ET-1-induced

proMMP-9 expression, cells were treated

with various concentration ET-1 for the

indicated time intervals. The condition

media were collected to assay proMMP-9

expression by gelatin zymography. (b) Time

dependence of ET-1-induced gene expres-

sion, cells were treated with 100 nM ET-1

for the indicated time intervals. MMP-9,

TIMP-1, and TIMP-2 mRNA were analyzed

by RT-PCR. (c) The MMP-9 mRNA was

also analyzed by real-time PCR. (d) The

cultured media were collected and analyzed

the level of MMP-9 protein by MMP-9 ELI-

SA kit. (e) ET-1-induced cell migration was

evaluated by migration assay. Cells were

pre-treated with MMP2/9 inhibitor (2/9i,

3 lM) for 1 h and then incubated with ET-1

(100 nM) for 48 h. The random phase

contrast images of RBA-1 cells were taken

(upper part, n = 3). The number of ET-1-

induced cell migration was counted as de-

scribed in ‘Materials and Methods’ (lower

part). Data are expressed as mean ± SEM

of three independent experiments.

*p < 0.05; #p < 0.01, as compared with

vehicle (a, b, c, d) or ET-1 alone (e). The

image represents one of at least three

individual experiments.



mRNA levels of MMP-9, TIMP-1, and TIMP-2 in ET-1-treated astrocytes were analyzed by RT-PCR. The resultsshowed that ET-1 induced an increase in MMP-9 expression,but not TIMP-1 and TIMP-2 in RBA-1 cells (Fig. 1b). Tofurther examine whether the increase of proMMP-9 expres-sion by ET-1 resulted from the induction of MMP-9 mRNAexpression, the real-time PCR analysis was performed. Thedata showed that ET-1 time-dependently induced MMP-9mRNA expression in RBA-1 cells (Fig. 1c). There was asignificant increase in MMP-9 mRNA within 6 h, reached amaximal response within 16 h, and sustained over 24 hduring the period of observation. We further evaluated theprotein level of MMP-9 in the culture medium using anELISA analysis. Data in Fig. 1(d) showed that the levels ofMMP-9 in the medium collected from the cells treatedwithout or with ET-1 for 16 and 24 h were 3.76 ± 1.02,7.96 ± 0.35, and 8.83 ± 1.74 pg/mL. These results sug-gested that ET-1-induced proMMP-9 expression occurred attranscriptional and translational levels. Moreover, to demon-strate the consequence of proMMP-9 expression induced byET-1, we evaluated its functional effect on RBA-1 cells by amigration assay. The images showed that ET-1 inducedRBA-1 cell migration after 48 h of incubation (Fig. 1e, upperpart). The number of migratory RBA-1 cells was summarizedas shown in Fig. 1(e). We found that ET-1-induced cellmigration was significantly blocked by pre-treatment withthe inhibitor of MMP-2/9 activity (MMP-2/9i, 3 lM)(Fig. 1e, lower part), suggesting that up-regulation ofproMMP-9 and its activity are required for enhancingRBA-1 cell migration induced by ET-1.

ET-1 induces proMMP-9 expression via Gi/o and Gq protein-coupled receptorsEndothelin-1 exerts its biological effects via the ET recep-tors, ETA and ETB, which are members of GPCR superfamily(Rubanyi and Polokoff 1994). Among the ET receptors, theETB receptor is predominantly expressed in astrocytes andmay mediate astrocytic hypertrophy in the injured CNS(Nakagomi et al. 2000; Rogers et al. 2003). We determinedthe expression of ETA and ETB receptors on RBA-1 cells bya real-time PCR analysis. As shown in Fig. 2(a), bothETA and ETB receptors were expressed in RBA-1 cells, andthe latter was expressed to a greater extent. Next, to identifythe subtypes of ET receptors responsible for ET-1-inducedproMMP-9 expression, cells were pre-treated with either BQ-123 (an ETA antagonist) or BQ-788 (an ETB antagonist)(IC50 = 0.78 ± 0.30 lM) (Saadoun and Garcıa 1999) for 1 hand then incubated with ET-1 for 16 h. These data showedthat selective blockade of ETB receptor, but not ETA receptor,inhibited the ET-1-induced proMMP-9 expression in RBA-1cells (Fig. 2b). Because ET receptors are characterized asGPCRs, we used G protein antagonists to determine thesubtypes of G proteins involved in ET-1-induced proMMP-9expression in RBA-1 cells. Pre-treatment with GP antago-

nist-2 (GPAnt2, a Gi/o protein antagonist) or GP antagonist-2A (GPAnt2A, a Gq protein antagonist) significantly inhib-ited ET-1-induced proMMP-9 expression (Fig. 2c). Thesedata indicated that ETB receptors coupled to Gi/o and Gq

proteins may predominantly mediate ET-1-induced proM-MP-9 expression in RBA-1 cells. To substantiate the role ofETB receptors in ET-1-induced functional response, weevaluated cell migration of RBA-1 cell using BQ-788. Asshown in Fig. 2(d), the images showed that ET-1-inducedcell migration was significantly attenuated by pre-treatmentwith 1 lM BQ-788, suggesting that ETB receptors mediatedthe cell migration induced by ET-1 in RBA-1 cells.

Furthermore, we investigated the ET-1-induced proM-MP-9 expression in primary culture of rat astrocytes. Thecharacteristics of primary cultured astrocytes were identifiedby immunofluorescence staining using an anti-GFAP anti-body (Fig. 2e). The zymographic data showed that theincreases in proMMP-9 expression induced by ET-1 weresignificantly reduced by pre-treatment with BQ-788,GPAnt2, or GPAnt2A (Fig. 2f), suggesting that ET-1 inducesproMMP-9 expression via ETB receptors coupled with Gi/o

and Gq proteins in primary culture of astrocytes.To confirm the contribution of ETB to MMP-9 induction,

we tested the effects of sarafotoxin 6C (S6C), an ETB

agonist, on ET-1-induced proMMP-9 expression in primaryastrocytes. Data in Fig. 2(g) showed that S6C inducedproMMP-9 expression in a time-dependent manner. Therewas a significant increase within 6 h and sustained over 24 h.In addition, blockades of ETB receptor and Gq proteininhibited the S6C-induced proMMP-9 expression in primaryastrocytes. However, blockade of Gi/o protein delayed theS6C-induced proMMP-9 expression as compared with that ofcontrol (Fig. 2g).

ET-1-induced proMMP-9 expression and cell migration ismediated through p42/p44 MAPK/Elk-1 cascadeAccumulating evidence has suggested that activation of p42/p44 MAPK by ET-1 modulates cellular functions of astro-cytes (Schinelli et al. 2001; He et al. 2007). To investigate therole of p42/p44 MAPK in ET-1-induced proMMP-9 expres-sion in RBA-1, cells were pre-treated with the inhibitor ofmitogen-activated protein kinase kinase 1/2 (MEK1/2)(U0126) for 1 h and then incubated with ET-1 for 16 h.These data showed that pre-treatment with U0126 attenuatedET-1-induced proMMP-9 expression in RBA-1 cells (Fig. 3a,left part). In addition, pre-treatment with U0126 concentra-tion-dependently inhibited ET-1-induced proMMP-9 expres-sion in primary culture of astrocytes (Fig. 3a, right part),suggesting that ET-1-stimulated p42/p44 MAPK is involvedin proMMP-9 induction in astrocytes.

Moreover, we determined whether phosphorylation ofp42/p44 MAPK was involved in the ET-1-induced responses,the kinetics of p42/p44 MAPK phosphorylation was assessedby western blotting using an anti-phospho-p42/p44 MAPK



antibody. The results showed that ET-1 stimulated p42/p44MAPK phosphorylation in a time-dependent manner andwith a maximal response within 3–5 min which wasinhibited by pre-treatment with 10 lM U0126 or 1 lMBQ-788 (Fig. 3b, upper part). Recent studies have suggestedthat phosphorylation of Ets family transcription factors maybe the consequence of activation of MAPK. Activation of Etsfamily members, particularly Elk-1, has been identified incerebral insults, and participates in production of inflamma-

tory mediators (Hu et al. 2004; Krupinski et al. 2005; Hsiehet al. 2008). Therefore, we investigated the role of transcrip-tion factor Elk-1 in ET-1-induced proMMP-9 expression inRBA-1 cells. The results showed that ET-1 stimulated a time-dependent phosphorylation of Elk-1 with a maximal responsewithin 10 min and sustained for 15 min during the period ofobservation in RBA-1 cells (Fig. 3b, lower part). To identifyrelated upstream components in these responses, cells werepre-treated with inhibitors of MEK1/2 (U0126, 10 lM) or

0.09

0.08

0.071/DC

t0.06

proMMP-9

GAPDH

8

6

proM

MP

-9 e

xpre

ssio

n(f

old

of b

asal

)

4

2

0

8

6

proM

MP

-9 e

xpre

ssio

n(f

old

of b

asal

)

4

2

0

8

** # #

## #

6

proM

MP

-9 e

xpre

ssio

n(f

old

of b

asal

)

4

2

0

100

75

50#

25

0Basal – BQ-788

ET-1/ 48 h

Mig

rato

ry c

ells

(no.

of

cells

)

BQ-123 (µM)ET-1 (h)

– 1–

– 0.01 0.116

1

8

6

proM

MP

-9 e

xpre

ssio

n(f

old

of b

asal

)

4

2

0

proMMP-9

GAPDH

proMMP-9

GAPDH

proMMP-9

GAPDH

ETA ETB

BQ-788 (µM)ET-1 (h)

– 1–

– 0.01 0.116

1

GPAnt2 (µM)ET-1 (h)

– 10–

– 0.1 116

10 GPAnt2 (µM)ET-1 (h)

– 10–

– 0.1 116

10

(a)

(b)

(c)

(d)

Fig. 2 Involvement of Gi and Gq proteins

coupling to ETB receptors in ET-1-induced

proMMP-9 expression in RBA-1 cells. (a)

Real-time PCR analysis of ET receptor

expression in RBA-1 cells. (b) The cells

were pre-treated with BQ-123 or BQ-788 for

1 h and then incubated with ET-1 for 16 h.

(c) RBA-1 cells were pre-treated with Gi

antagonist (GPAnt2) or Gq antagonist

(GPAnt2A) for 1 h and then exposed to ET-

1 for the indication time intervals. (d) For

cell migration, cells were pre-treated with

BQ-788 for 1 h and then incubated with ET-

1 (100 nM) for 48 h. The random phase

contrast images of RBA-1 cells were taken

(upper part, n = 3). The number of ET-1-

induced cell migration was counted as de-

scribed in ‘Materials and Methods’. (e) The

primary rat brain culture of astrocytes

(RBA) were prepared, cultured and ana-

lyzed by immunofluorescence staining

using an anti-GFAP antibody. Moreover,

cells were pre-treated with BQ-788,

GPAnt2, or GPAnt2A for 1 h and then ex-

posed to ET-1 for the indicated time inter-

vals. The proMMP-9 protein was analyzed

by gelatin zymography (f). (g) ETB agonist

(S6C) induced proMMP-9 expression in

primary astrocytes. Data are expressed as

mean ± SEM of three individual experi-

ments. *p < 0.05; #p < 0.01, as compared

with the respective values of cells stimu-

lated by ET-1 alone. The figure represents

one of three individual experiments.



ETB receptor (BQ-788, 1 lM) for 1 h and then stimulated byET-1 for the indicated time intervals. The results showed thatpre-treatment with U0126 or BQ-788 blocked ET-1-stimu-lated Elk-1 phosphorylation (Fig. 3b, lower part), suggestingthat ETB-mediated p42/p44 MAPK activation is crucial forElk-1 activation in RBA-1 cells. To further ensure the role ofp42/p44 MAPK in ET-1-induced proMMP-9 expression,cells were transfected with shRNA for p42 MAPK. Asshown in Fig. 3(c), transfection with p42 shRNA knock-downed the expression of total p42 protein and attenuatedET-1-induced proMMP-9 expression, demonstrating thatp42/p44 MAPK was essential for ET-1-induced proMMP-9expression in RBA-1 cells.

To examine the functional response of ET-1-stimulatedp42/p44 MAPK activation, we evaluated cell migration ofRBA-1 cells. As shown in Fig. 3(d), the images showedthat ET-1-induced cell migration was significantly attenu-ated by pre-treatment with 10 lM U0126, suggesting thatp42/p44 MAPK participated in the cell migration inducedby ET-1 in RBA-1 cells. These results demonstrated thatp42/p44 MAPK/Elk-1 cascade was involved in ET-1-stimulated proMMP-9 expression and migration of RBA-1cells.

ET-1 induces cell migration via NF-jB-dependentproMMP-9 inductionAs shown in Fig. 1(c), ET-1 induced proMMP-9 expressionat transcriptional level. The promoter region of MMP-9possesses serial binding elements for recognition of tran-scription factors including NF-jB and AP-1 (Yong et al.2001; Rosenberg 2002). The NF-jB family is considered asan essential regulator of cellular activities associated withinflammation (Ghosh and Hayden 2008). ET-1 has beenshown to stimulate NF-jB activation, which modulatescellular functions in various cell types (Morigi et al. 2006;Banerjee et al. 2007). Moreover, MMP-9 promoter containsthe jB binding site that is crucial for induction of MMP-9(Rosenberg 2002; Hsieh et al. 2008). Thus, we examinedwhether activation of NF-jB was required for proMMP-9expression induced by ET-1 in RBA-1 cells. First, cells werepre-treated with a selective NF-jB inhibitor, Bay11-7082,which blocks activation of NF-jB signaling (Huang et al.2002), and then incubated with ET-1 for 16 h. The zymo-graphic data showed that pre-treatment with Bay11-7082concentration-dependently reduced ET-1-induced proMMP-9expression in RBA-1 cells (Fig. 4a, left part) and primaryculture of astrocytes (Fig. 4a, right part), suggesting the

proMMP-9

proMMP-2

proMMP-9

proMMP-2

proMMP-9

proMMP-2

proMMP-9

proMMP-2

proMMP-9

proMMP-2

Inhibitor

S6C (h)

InhibitorET-1 (h) 0 16 24

–0 16

BQ-788 (1 µM)24

0 6 16 24

–

Inhibitor

S6C (h) 0 6 16 24

GPAnt2

Inhibitor

S6C (h) 0 6 16 24

GPAnt2A

Inhibitor

S6C (h) 0 6 16 24

BQ-788

Inhibitor

S6C (h) 0 6 16 24

U0126

Fold of basal

proMMP-9

proMMP-2

Fold of basal

proMMP-9

proMMP-2

Fold of basal

proMMP-9

proMMP-2

1.0 3.2 3.5 2.0 1.5# 1.0#

1.0 4.2 3.3 2.7 2.6* 2.6#

1.0 3.4 3.6 1.4 1.6# 1.3#


–0 16

GPAnt2 (10 µM)24


–0 16

GPAnt2A (10 µM)

24

(f)

(g)(e)

Fig. 2 (Continued ).



involvement of NF-jB in ET-1-induced proMMP-9 expres-sion. To further verify that NF-jB is important in ET-1-induced proMMP-9 expression, cells were transfected withp65 siRNA and then stimulated by ET-1 for 16 h. As shownin Fig. 4(b), transfection with p65 siRNA knock-downed theexpression of p65 NF-jB protein and attenuated the ET-1-induced proMMP-9 expression, whereas a housekeepingprotein GAPDH was not changed. These data suggested thatp65 NF-jB was involved in proMMP-9 induction by ET-1.Moreover, cell migration assay was performed to determinewhether NF-jB was involved in ET-1-induced RBA-1migration. The images showed that ET-1-induced cell

migration was significantly inhibited by pre-treatment with1 lM Bay11-7082 (Fig. 4c). These data demonstrated thatNF-jB is necessary for ET-1-induced proMMP-9 expressionand cell migration in RBA-1 cells.

The NF-jB translocation from cytosolic fraction intonucleus is an effective index in reflecting the state ofNF-jB activation. Thus, we determined whether ET-1stimulated the translocation of NF-jB (p65 subunit) byimmunofluorescent staining. As shown in Fig. 5(a), theimaging data showed that ET-1 stimulated translocationof p65 NF-jB into nucleus with a maximal responsewithin 60 min and sustained over 120 min, which was

proMMp-9

GAPDH

8

# # #

6

proM

MP

-9 e

xpre

ssio

n(f

old

of b

asal

)

4

2

0U0126 (µM)

ET-1 (h)–

–

P-p44

P-Elk-1

proMMP-9p42

– pTopo sh-p42

P-p42

GAPDH

GAPDH

GAPDHGAPDH

Inhibitor –0 1 3 5 0 1 3 5 0 1 3 5

BQ-788U0126ET-1 (min)

0.1 116

1010 –

Fold of basal 1.0 2.4 2.4 1.3 1.4* 1.8*

proMMP-9

proMMP-2

InhibitorET-1 (h) 0 16 24 0 16

U0126 (10 µM)24

–

Inhibitor

shRNAET-1 (h)

––

–16

pTopo sh-p42 pTopo sh-p42

Basal

125

100

75

50

25

0#M

igra

tory

cel

ls(n

o. o

f ce

lls)

– U0126

ET-1/ 48 h

–0 5 10 15 0 5 10 15 0 5 10 15

BQ-788U0126ET-1 (min)

(a)

(b)

(c)

(d)

Fig. 3 Involvement of p42/p44 MAPK/Elk-1

cascade in ET-1-induced proMMP-9

expression in RBA-1 cells. (a) Cells, RBA-1

cell line (left part) or primary culture of RBA

(right part), were treated with 100 nM ET-1

for 16 h in the absence or presence of

U0126. ProMMP-9 expression was deter-

mined by zymography as described in

Fig. 1. (b) Time dependence of ET-1-stim-

ulated p42/p44 MAPK and Elk-1 phos-

phorylation, cells were incubated with

100 nM ET-1 for the indicated times in the

absence or presence of U0126 (10 lM) or

BQ-788 (1 lM). (c) Cells were transfected

with p42 shRNA for 24 h and then exposed

to ET-1 for 16 h. The condition media and

cell lysates were collected and analyzed by

gelatin zymography and western blotting

using an anti-phospho-p42/p44 MAPK, anti-

phospho-Elk-1, anti-p42, anti-Elk-1, or anti-

GAPDH (as an internal control) antibody.

(d) For cell migration, cells were pre-treated

with 10 lM U0126 for 1 h and then incu-

bated without (Basal) or with ET-1 (100 nM)

for 48 h. Data are expressed as mean ±

SEM of at least three individual experi-

ments. *p < 0.05; #p < 0.01, as compared

with ET-1 alone. The figure represents one

of at least three individual experiments.



significantly attenuated by pre-treatment with the inhibitorsof NF-jB (Bay11-7082, 1 lM), ETB receptors (BQ-788,1 lM) or MEK1/2 (U0126, 10 lM) (Fig. 5b). To furtherdetermine whether the activation of NF-jB signaling wasmediated through phosphorylation of IKKa/b, the phos-phorylation of IKKa/b was determined by western blotusing an anti-phospho-IKKa/b antibody. As shown inFig. 5(c), ET-1 stimulated phosphorylation of IKKa/b in atime-dependent manner, which was inhibited by pre-treat-ment with Bay11-7082, BQ-788, or U0126. Collectively,these results revealed that ET-1-activated IKKa/b/NF-jBcascade participated in proMMP-9 induction and cellmigration in RBA-1 cells.

To further ensure the involvement of IKKa/b in ET-1-induced proMMP-9 expression, cells were transfected with adominant negative IKKa/b mutant (DIKKa/b) and thenincubated with ET-1 for 16 h. As shown in Fig. 5(d), theprotein level of IKKa/b was increased by transfection withDIKKa/b, which attenuated the ET-1-induced proMMP-9expression. These data suggested that IKKa/b was involvedin proMMP-9 induction by ET-1. These results indicated that

ET-1 activated the IKKa/b/NF-jB cascade through ETB andp42/p44 MAPK signaling in RBA-1 cells.

ET-1 up-regulates proMMP-9 expression via AP-1activationRecent studies suggest that the binding elements of theMMP-9 promoter region include two AP-1 sites, which arerequired for MMP-9 induction (Wang et al. 2009; Wu et al.2009). To examine whether AP-1 contributed to ET-1-induced proMMP-9 expression, cells were pre-treated withtanshinone IIA (TSIIA, an inhibitor of AP-1) for 1 h and thenincubated with ET-1 for 16 h. These data revealed that ET-1-induced proMMP-9 expression was blocked by pre-treatmentwith TSIIA in RBA-1 cells (Fig. 6a, left part) and primaryculture of astrocytes (Fig. 6a, right part). These resultsindicated that the transcription factor AP-1 may contribute toinduction of proMMP-9 by ET-1.

We further examined whether ET-1 affected c-Jun phos-phorylation and c-Fos expression in RBA-1 cells. First, ET-1-stimulated c-Jun phosphorylation was analyzed by westernblotting using an anti-phospho-c-Jun antibody. As expected,

proMMP-9

GAPDHFold of basal 1.0 2.4 3.1 1.7 1.2* 1.5*

proMMP-9

proMMP-2

Fold of basal 1.0 1.4 1.6 8.9 8.1 6.3*

proMMP-9

p65

scrb si-p65–

GAPDHGAPDH

10

* *

8

proM

MP

-9 e

xpre

ssio

n(f

old

of b

asal

)

6

4

2

125

100

75

Mig

rato

ry c

ells

(no

. of

cells

)

50

25

0Basal – Bay

#

ET-1/ 48 h

0Bay11-7082 (µM)

Inhibitor

ET-1 (h) 0 16 24 0 16 24

Bay11-7082 (1 µM)–

–

–

0.01

16

0.01 0.1 1–

ET-1 (h)

siRNA –

–

scrb si-p65 scrb si-p65

16

–

ET-1 (h)

(a)

(b)

(c)

Fig. 4 NF-jB (p65) is essential for ET1-1-

induced proMMP-9 expression in RBA-1

cells. (a) Cells, RBA-1 cell line (left part) or

primary RBA (right part), were treated with

100 nM ET-1 for 16 h in the absence or

presence of Bay11-7082 (0.01, 0.1 or

1 lM). ProMMP-9 expression was deter-

mined by zymography as described in

Fig. 1. (b) Cells were transfected with

scramble (scrb) or p65 siRNA for 24 h and

then exposed to ET-1 for 16 h. The condi-

tion media and cell lysates were collected

and analyzed by zymography for proMMP-9

and western blotting for p65 and GAPDH

(as an internal control), as described in

Methods. (c) For cell migration, cells were

pre-treated with 1 lM Bay11-7082 for 1 h

and incubated without (Basal) or with ET-1

(100 nM) for 48 h. Data are expressed as

mean ± SEM of at least three independent

experiments. *p < 0.05; #p < 0.01, as com-

pared with ET-1 alone. The figure repre-

sents one of at least three individual

experiments.



ET-1 stimulated c-Jun phosphorylation in a time-dependentmanner, which was significantly inhibited by pre-treatmentwith TSIIA and BQ-788 during the period of observation(Fig. 6b). To examine whether ET-1 also induced immediateearly gene c-Fos/AP-1 expression, RT-PCR analysis wasperformed. As shown in Fig. 6(c), ET-1-induced c-fosmRNA expression was significantly inhibited by pre-treat-ment with TSIIA or BQ-788. These results demonstratedthat ET-1 induced c-Jun phosphorylation and c-fos expres-sion through ETB receptors and AP-1. To further verify theroles of c-Jun and c-Fos in ET-1-induced proMMP-9expression, cells were transfected with shRNA for c-Junor c-Fos and then incubated with ET-1 for 16 h. The datashowed that transfection with siRNA for c-Jun and c-Fos

significantly knock-downed the expression of c-Jun (Fig. 6e,right part) and c-Fos (Fig. 6f, right part), and reduced theET-1-induced proMMP-9 expression (Fig. 6e and f). Thesedata suggested that c-Jun and c-Fos are involved inproMMP-9 induction by ET-1. Next, cell migration assaywas performed to determine the role of AP-1 in ET-1-induced proMMP-9 expression associated with cell migra-tion. The images showed that ET-1-induced cell migra-tion was significantly inhibited by pre-treatment with 10 lMTSIIA (Fig. 6d), suggesting that AP-1 is involved in theET-1-induced cell migration. These results indicated thatAP-1 (i.e. c-Jun and c-Fos) activity was required for ET-1-induced proMMP-9 expression and cell migration in RBA-1cells.

P-IKKαα/β

GAPDH

Inhibitor

ET-1 (min) 0 10 30

–

45 60 120

P-IKKα/β

GAPDH

Inhibitor

ET-1 (min) 0 10 30

Bay11-7082

45 60 120

P-IKKα/β

GAPDH

GAPDH

Plasmid –

–

pcDNA3

16

–ΔIKKα/β pcDNA3 ΔIKKα/βET-1 (h)

proMMp-9

Inhibitor

ET-1 (min) 0 10 30

BQ-788

45 60 120

P-IKKα/β

GAPDH

IKKα/β

GAPDH

pcDNA3 ΔIKKα/β–

Inhibitor

ET-1 (min) 0 10 30

U0126

45 60 120

(a)

(b)

(c)

(d)

Fig. 5 ET-1 induces proMMP-9 expression

via an IKK/NF-jB pathway in RBA-1 cells.

(a) Time dependence of ET-1-induced NF-

jB p65 subunit translocation. (b) Cells were

treated with 100 nM ET-1 for 60 min in the

absence or presence of Bay11-7082

(1 lM), BQ-788 (1 lM) or U0126 (10 lM).

The p65 NF-jB translocation was deter-

mined by an immunofluorescence staining

using anti-p65 antibody. (c) Time depen-

dence of ET-1-stimulated IKKa/b phos-

phorylation, cells were incubated with

100 nM ET-1 for the indicated time intervals

in the absence or presence of Bay11-7082,

BQ-788 or U0126. The IKKa/b phosphory-

lation were determined by western blot as

described in Methods. (d) Cells were

transfected with an empty vector (pcDNA3)

or dominant negative IKK mutants (DIKKa/

b) for 24 h and then exposed to ET-1 for

16 h. The condition media and cell lysates

were collected and analyzed by gelatin

zymography for proMMP-9 and western

blotting for IKKa/b phosphorylation and

GAPDH (as an internal control). The figure

represents one of at least three individual

experiments.



Involvement of binding elements in the activation of therat MMP-9 gene promoter by ET-1We have found that ET-1 stimulates activation of varioustranscription factors including Elk-1, NF-jB, and AP-1 inRBA-1 cells. Next, we examined whether the binding ofthese transcription factors to their promoter binding elementswas essential for ET-1-induced MMP-9 gene regulation. Therat MMP-9 promoter luciferase reporter was constructed andits activity was evaluated by promoter-luciferase activityassay. The rat MMP-9 promoter was constructed into apGL3-basic vector containing a luciferase reporter system (asillustrated in Fig. 7a, upper part; pGL-MMP-9-Luc), which

contains several putative recognition elements for a variety oftranscription factors that include NF-jB, Ets, and AP-1families. Thus, to determine the effect of ET-1 on the MMP-9promoter activity, cells were transfected with a pGL-MMP-9-Luc construct and then incubated with ET-1 (100 nM) for theindicated time intervals. As shown in Fig. 7(a), ET-1increased the MMP-9 promoter activity in a time-dependentmanner. A maximal response was obtained within 16 h,which was significantly inhibited by pre-treatment with anETB antagonist (BQ-788, 1 lM) and the inhibitors of MEK1/2 (U0126, 10 lM), NF-jB (Bay11-7082, 1 lM), or AP-1(TSIIA, 10 lM) (Fig. 7b). To further ensure that Elk-1,

proMMP-9

GAPDH

8Fold of basal 1.0 4.3 4.9 1.5 2.5* 2.3*

proMMP-9

proMMP-2

Inhibitor

ET-1 (h)

Inhibitor –

ET-1 (min) 0 30 60 120 0 30 60

90Inhibitor

ET-1 (min)

– TSIIA

30

BQ-788

–

60

Mig

rato

ry c

ells

(no

. of

cells

)30

0

proMMP-9

GAPDH

shRNA

ET-1 (h)

–

–

pTopo Sh-c-Jun –

16

pTopo Sh-c-Jun

Basal – TSIIA

#

ET-1/ 48 h

pTopo Sh-c-Jun

c-Jun

–

pTopo Sh-c-Jun–

120 0 30 60 120

BQ-788TSIIA

0 16 24 16 24

TSIIA (10 µM)

0

–

* #

#

6

proM

MP

-9 e

xpre

ssio

n(f

old

of b

asal

)

4

2

0TSIIA (µM)

ET-1 (h)

P-c-Jun

GAPDH

GAPDH

c-Fos

GAPDH

proMMP-9

GAPDH

c-Fos

b-actin

–

–

10 – 0.1

16

1 10

shRNA

ET-1 (h)

–

–

pTopo Sh-c-Jun –

16

pTopo Sh-c-Jun

(d)

(e)

(f)

(a)

(b)

(c)

Fig. 6 AP-1 is crucial for ET-1-induced

proMMP-9 expression in RBA-1 cells. (a)

Cells, RBA-1 cell line (left part) or primary

culture of RBA (right part), were treated with

100 nM ET-1 for 16 h in the absence or

presence of tanshinone IIA (TSIIA). ProM-

MP-9 expression was determined by gelatin

zymography. (b) Cells were pre-treated with

10 lM TSIIA or 1 lM BQ-788 for 1 h and

then exposed to ET-1 for the indicated time

intervals. The cell lysates were analyzed by

western blotting using an anti-phospho-c-

Jun antibody or anti-GAPDH (as an internal

control). (c) Cells were pre-treated with

10 lM TSIIA or 1 lM BQ-788 for 1 h and

then exposed to ET-1 for the indicated

times. RNA was extracted and analyzed by

RT-PCR to determine c-Fos gene expres-

sion. To ensure the involvement of c-Jun

and c-Fos in ET-1-induced proMMP-9

expression, cells were transfected with (e)

c-Jun or (f) c-Fos shRNA for 24 h and then

exposed to ET-1 for 16 h. ProMMP-9

expression was determined by gelatin zy-

mography. (d) For cell migration, cells were

pre-treated with 10 lM TSIIA for 1 h and

then incubated without (Basal) or with ET-1

(100 nM) for 48 h. Data are expressed as

mean ± SEM of at least three independent

experiments. *p < 0.05; #p < 0.01, as com-

pared with ET-1 alone. The figure repre-

sents one of at least three individual

experiments.



NF-jB, and AP-1 indeed mediated ET-1-induced MMP-9promoter activity through binding to their regulatory ele-ments within the MMP-9 promoter region, the wild-type(WT) MMP-9 promoter mutated by a single-point mutationof the Ets binding site (mt-Ets-MMP-9), the jB binding site(mt-jB-MMP-9), and the AP-1 binding site (mt-AP-1-MMP-9) were constructed (as indicated in Fig. 7c, insert panel),ET-1 stimulated MMP-9 promoter activity was significantlyattenuated in RBA-1 cells transfected with mt-Ets-MMP-9,

mt-jB-MMP-9, or mt-AP-1-MMP-9, indicating that Ets, jB,and AP-1 elements were essential for ET-1-induced MMP-9promoter activity. These results further confirmed that ET-1induced MMP-9 promoter activity via enhancing Elk-1, NF-jB, and AP-1 binding to the Ets, jB, and AP-1 elements ofthe MMP-9 promoter, respectively, in RBA-1 cells.

Furthermore, we determined the roles of ET-1 receptorsand related transcription factors in ET-1-induced MMP-9gene expression. RBA-1 cells were pre-treated with GPAnt2

3

ET-1 (16 h) – –– –

––

––

–

– – – –

–

–––

––

––

–

–––

––

–

++ +

+

+ + +

++

++

+WT-MMP-9

2

* *

#1

0

Rel

ativ

e lu

cife

rase

act

ivit

y(f

old

of b

asal

)

mt-Ets-MMP-9

mt-κB-MMP-9

mt-AP-1-MMP-9

mt-Ets-MMP-9

mt-κB-MMP-9

mt-AP-1-MMP-9

Luc

Luc

Luc

EtsκB

κB

κB

AP-1 AP-1

Ets AP-1 AP-1+1

+1

+1

Ets AP-1 AP-1

WB: MMP-9(medium)

RT-PCR: b-actin(RNA extracts)

— —Basal GPAnt2 GPAnt2A BQ-788

ET-1/ 16 h

U0126 Bay TSIIA

Rel

ativ

e lu

cife

rase

act

ivit

y(f

old

of b

asal

)

15

10

5

0Basal GPAnt2— GPAnt2A BQ-788

ET-1/ 16 h

* **

*

# #

U0126 Bay TSIIA

pGL-MMP-9-Luc

3

2

1

0

Rel

ativ

e lu

cife

rase

act

ivit

y(f

old

of b

asal

)

0 2

ET-1, time (h)

4 6 16 24

WT-MMP-9κB Ets AP-1

+1

AP-1

#

##

Luc3

2

1

Inhibitor

16 h

BQ-788 U0126 Bay TSIIA—

—ET-1

0

Rel

ativ

e lu

cife

rase

act

ivit

y(f

old

of b

asal

)

pro MMP-9

## #

#

(a)(b)

(c) (d)

(e)

Fig. 7 ET-1-stimulated MMP-9 promoter activity, mRNA expression

and protein secretion is mediated through Elk-1/NF-jB/AP-1 path-

ways in RBA-1 cells. (a) Schematic representation of a 5¢-promoter

regions of the rat MMP-9 gene fused to the pGL-luciferase reporter

gene (pGL-MMP-9-Luc), the translational start site (+1) of the lucif-

erase reporter gene was indicated by an arrow. RBA-1 cells were

transiently co-transfected with pGL-MMP9-Luc and pGal encoding for

b-galactosidase for 24 h. The cells were treated with or without ET-1

(100 nM) for the indicated time intervals. (b) After co-transfection,

cells were pre-treated with BQ-788 (1 lM), U0126 (10 lM), Bay11-

7082 (Bay, 1 lM) or TSIIA (10 lM) for 1 h, and then incubated with

ET-1 for 16 h. (c) Activation of wild-type (WT), Ets-(mt-Ets), jB-(mt-

jB), or AP-1-point-mutated (mt-AP-1) MMP-9 promoter constructs by

ET-1. Schematic representation of the different MMP-9-luciferase

constructs, either wild-type (WT) or modified by single-point mutation

of the Ets, jB, or AP-1 binding site (inset panel). After co-transfection

for 24 h, promoter activities of different MMP-9-promoter constructs

stimulated with or without ET-1 (100 nM) for 16 h, were measured as

relative MMP-9 promoter activity to that of b-galactosidase. The rel-

ative increase in MMP-9 promoter activity induced by ET-1 normal-

ized to un-stimulated cells is indicated as fold increase. Cells were

pre-treated with GPAnt2 (10 lM), GPAnt2A (10 lM), BQ-788 (1 lM),

U0126 (10 lM), Bay11-7082 (Bay, 1 lM), or TSIIA (10 lM) for 1 h,

and then incubated with 100 nM ET-1 for 16 h. The expression of

MMP-9 mRNA induced by ET-1 was analyzed by real-time PCR (d).

Condition media were analyzed by western blotting using an anti-

MMP-9 antibody (e). Data are expressed as mean ± SEM of at least

three independent experiments. *p < 0.05; #p < 0.01, as compared

with ET-1 alone. The figure represents one of at least three individual

experiments.



(10 lM), GPAnt2A (10 lM), BQ-788 (1 lM), U0126(10 lM), Bay11-7082 (1 lM), or TSIIA (10 lM) for 1 hand then incubated with ET-1 (100 nM) for 16 h. Pre-treatment with these inhibitors significantly attenuated ET-1-induced MMP-9 mRNA (Fig. 7d) and protein (Fig. 7e)expression, suggesting that ET-1 induces MMP-9 geneexpression via ETB receptors linking to activation of p42/p44 MAPK/Elk-1, NF-jB, AP-1 cascades in RBA-1 cells.

Discussion

The MMP-9 activity has been implicated in neuroinflamma-tion, which involves BBB disruption and cell death in theCNS (Yong et al. 2001; Rosenberg 2002). It is reported thatelevated MMP-9 activity contributes to the progression ofCNS pathology (Aoki et al. 2002; Wang et al. 2002; Harriset al. 2007). Using pharmacological blockades or geneknock-out strategies, prevention of MMP-9 activity exhibitsprotective effects on the brain after cerebral ischemia (Svedinet al. 2007; McColl et al. 2008). In the CNS, ischemic injuryelicits ET-1 production from astrocytes (Hasselblatt et al.2001), related to the destructive outcomes of ischemic braininjury (Lo et al. 2005). The physiopathological effects of ET-1 on astrocytes are substantially mediated through ETB

receptors (Nakagomi et al. 2000; Rogers et al. 2003).However, the role of ET-1 activity on astrocytic functionsin the CNS diseases remains elusive. Here, we use culturedmodels of rat astroglial cell line (RBA-1) and primary ratbrain culture of astrocytes to investigate the mechanismsunderlying ET-1-induced MMP-9 expression and functionalchanges. These results suggest that in rat astrocytes, activa-tion of ETB receptor-dependent p42/p44 MAPK/Elk-1,NF-jB, and AP-1 signaling cascades is essential for ET-1-induced MMP-9 gene expression and cell migration.

In addition to maintain the functional homeostasis in CNSmicroenvironment, astrocytes have been demonstrated toparticipate in the regulation of cerebral blood flow. Itrepresents control mechanisms that match oxygen andglucose delivery through blood flow with the local metabolicdemands that are imposed by neural activity (Iadecola andNedergaard 2007). As a fundamental component of theneurovascular unit, recent work has revealed the roleof astrocyte dysfunction in neurodegenerative diseases(Iadecola 2004; Guo and Lo 2009). Thus, we study howET-1 modulates astrocytic functions.

First, we demonstrated that ET-1-triggered activation ofsignaling molecules is mediated via ETB receptor in ratastrocytes. By real-time PCR, zymographic, and migrationanalyses, we found that activation of ETB receptor coupled toGi/o and Gq proteins was essential for ET-1-induced proM-MP-9 expression in rat astrocytes (Fig. 2). These data wereconsistent with the findings that activation of ETB receptor isresponsible for characteristics of reactive gliosis (Rogerset al. 2003; Gadea et al. 2008) and of resistance artery

remodeling in diabetes (Sachidanandam et al. 2007). How-ever, in respiratory and cardiovascular systems, both ETreceptor subtypes, ETA in particular, are involved inprogression of airway diseases (De Lagausie et al. 2005;Lund et al. 2009). The data from real-time PCR showed apredominant expression of ETB receptor in RBA-1 cells(Fig. 2a). As there was no statistical significance of ETA-selective antagonist BQ-123 on ET-1-induced proMMP-9expression (Fig. 2b), we suggested that ETB receptor playeda more important role than ETA receptor in ET-1-inducedMMP-9 induction, consistent with the significance of ETB

receptor in the modulation of astrocytic hypertrophy in theinjured CNS (Nakagomi et al. 2000; Rogers et al. 2003).

Within the MMP-9 promoter, there are numerous tran-scription factor binding sites, which control MMP-9 geneexpression. Additionally, increasing studies have demon-strated that diverse extracellular stimuli, such as IL-1b,bradykinin (BK), and oxidized low-density lipoprotein(oxLDL), prompt MMP-9 induction through Elk-1 (amember of Ets family), NF-jB, and AP-1, respectively(Wu et al. 2004; Hsieh et al. 2008; Wang et al. 2009, 2010).These extensive researches indicate that Ets, NF-jB, and AP-1 may regulate MMP-9 expression through direct, indirect(e.g., induction of other transcription factors such as c-Fos orc-Jun), or synergistic action. Moreover, a current study hasidentified that ET-1 induces MMP-9 expression via NF-jB inhuman osteosarcoma (Felx et al. 2006). However, very fewdata indicate that ET-1 regulates MMP-9 through AP-1 or Etsfamily. In this study, we demonstrated that ET-1-inducedMMP-9 expression is mediated through NF-jB, AP-1, andElk-1 transcription factors in RBA-1 cells. All these threetranscription factors may be essential for MMP-9 up-regulation by ET-1.

Several lines of evidence have suggested that activation ofp42/p44 MAPK by ET-1 regulates cellular functions ofastrocytes (Schinelli et al. 2001; He et al. 2007). Activationof MAPK/Elk-1 pathway has also been shown to be involvedin MMP-9 induction in different cell types (Tanimura et al.2002; Hsieh et al. 2008). We further demonstrated that ET-1stimulated an ETB receptor-dependent cascade of sequentialp42/p44 MAPK/Elk-1 phosphorylation (Fig. 3b), whichcontributes to induction of MMP-9 promoter activity(Fig. 7b and c), mRNA, and protein levels (Fig. 7d and e).These results were consistent with those of obtained withBK- and oxLDL-induced MMP-9 expression in RBA-1 cells(Hsieh et al. 2008; Wang et al. 2010).

The transcription factor NF-jB is a ubiquitous regulator ofgene expression associated with inflammatory responses. Ourresults revealed that IKKa/b and NF-jB activation wererequired for ET-1-induced proMMP-9 expression by blockadeof NF-jB signaling using the pharmacological and transfec-tion strategies (Figs 4 and 5). It has been confirmed that NF-jB signaling is required for MMP-9 induction both in vitroand in vivo (Hsieh et al. 2004; Tai et al. 2008), which



enhances cell motility (Hsieh et al. 2008) and tumor invasion(Tai et al. 2008). The mechanism of NF-jB activationstimulated by ET-1 remains elusive. Our data suggested thatETB-mediated p42/p44 MAPK activation promoted IKKa/bphosphorylation (Fig. 5c) and NF-jB translocation (Fig. 5b)in RBA-1 cells. In human U87 astrocytoma cells, p42/p44MAPK has been suggested to be necessary for IKKphosphorylation (Kam et al. 2007). Besides, another MAPKmember p38 MAPK, has been demonstrated to mediate tumornecrosis factor (TNF)-a-triggered IKK activation in skeletalmuscle (De Alvaro et al. 2004). These findings imply that indifferent cell types, distinct MAPK members are potentiallyassociated with IKK phosphorylation. We further providedevidence to ensure that ET-1-triggered NF-jB activity wasobligatory for MMP-9 induction. We found that pre-treatmentwith Bay11-7082 attenuated ET-1-stimulated MMP-9 pro-moter activity (Fig. 7b), mRNA, and protein levels (Fig. 7dand e), as well as mutation of the NF-jB element retardedET-1-induced MMP-9 promoter activity (Fig. 7c). These datawere consistent with the mechanisms of MMP-9 expression inthe human breast cancer cell line MCF-7 (Tai et al. 2008;Park et al. 2009).

The results from Fig. 5(a) showed that ET-1 stimulatedtranslocation of p65 NF-jB at 30 min and sustained over120 min. Moreover, the rapid activation of NF-jB isessential for MMP-9 induction in astrocytes (Hsieh et al.2004). According to these results, we considered that ET-1-mediated responses, including NF-jB activation, proMMP-9mRNA and protein expression occur in a lineage relation-ship. ET-1 has been shown to induce IL-1b release inastrocytes (Didier et al. 2003). Moreover, IL-1b alsopromotes MMP-9 expression in rat brain astrocytes (Wuet al. 2004). These reports further support that otherpossible mechanisms, such as secretion of inflammatorycytokines, may be involved in ET-1-induced proMMP-9expression.

Accumulating evidence indicates that MMP-9 is producedvia AP-1-dependent mechanisms in various cell types (Byunet al. 2006; Wang et al. 2009). AP-1 family proteins areimportant transcription factors that regulate the MMP-9 geneexpression by different stimuli (Xu et al. 2001; Woo et al.2004). c-Jun/c-Fos has been shown to be one of the well-characterized examples of AP-1 family. Upon stimulation,c-Jun could dimerize with c-Fos to form stable heterodimersthat bind to a specific AP-1 site in the promoter region oftarget genes and enhances the gene transcription (Shaulianand Karin 2001). Therefore, we investigated the role ofc-Jun and c-Fos in ET-1-induced proMMP-9 expression. Ourdata showed that c-Jun phosphorylation and c-Fos up-regulation were essential for ET-1-induced proMMP-9expression (Fig. 6), as well as MMP-9 promoter activity(Fig. 7b), mRNA and protein levels (Fig. 7d and e). Suchinhibitory effects were also achieved by mutation of the AP-1 element on MMP-9 promoter activity (Fig. 7c). The data

were consistent with the mechanisms of MMP-9 expressionin human breast cancer cells (Byun et al. 2006) andastrocytes (Wang et al. 2009).

Cell motility is a vital process involved in embryonicdevelopment, wound healing, inflammatory responses, andtumor metastasis (Lauffenburger and Horwitz 1996). Recentstudies further suggest the correlation between MMP-9, cellmigration, and glial scar formation (Takenaga and Kozlova2006; Hsu et al. 2008), implying that MMP-9 may beinvolved in brain remodeling after injury. It has been reportedthat p42/p44 MAPK/Elk-1, NF-jB, and AP-1 are involved inMMP-9 up-regulation, which is crucial for regulating cellmotility in different cell types (Byun et al. 2006; Hsieh et al.2008; Mantuano et al. 2008; Weng et al. 2008; Wang et al.2009). In this study, we demonstrated that ET-1 induced cellmigration required MMP-9 activity (Fig. 1c) via coordina-tion of transcription factors including Elk-1, NF-jB, andAP-1 (Figs 3d, 4c, and 6d). To rule out the possibility of cellproliferation in ET-1-induced cell migration, hydroxyurea, aninhibitor of DNA synthesis (Yarbro 1992), was used toprevent proliferation of astrocytes during the period ofobservation in the migration assay. Therefore, we furthersuggested that up-regulation of proMMP-9 by ET-1 isessential for enhancing RBA-1 migration. Moreover, wefound that the pharmacological studies showed ET-1-inducedMMP-9 expression is only partially blocked by NF-jBinhibitor Bay11-7082 and AP-1 inhibitor TSIIA. On theother hand, ET-1-induced RBA-1 cell migration is com-pletely blocked by Bay11-7082 and TSIIA. We consideredthat the discrepancy is because of the different experimentalconditions, or ET-1-mediated cell migration may requireother proteins besides MMP-9.

Astrocytic migrationET-1

ETBPlasma membrane

Nuclear membrane

mmp-9AP-1EtsκB

proMMP-9

βγ

Gαq

GI/O

NF-κB

NF-κB

IKKα/β MEK1/2

p42/p44MAPK

EIk-1

PP Jun Fos

Fig. 8 Schematic representation of signaling pathways for ET-1-in-

duced proMMP-9 expression and cell migration in astrocytes. ET-1

functionally regulates proMMP-9 expression and cell migration via ETB

receptor-dependent p42/p44 MAPK/Elk-1, NF-jB, and AP-1 signaling

cascades in astrocytes.



Based on the typical characteristics of astrogliosis, theability of ET-1 to promote DNA synthesis and hypertrophyhas been identified in astrocyte models (Cazaubon et al.1997; Rogers et al. 2003). In endothelial cells, ET-1 has beenshown to prompt ETB-mediated migration, which is ulti-mately correlated with angiogenesis (Daher et al. 2008).However, ET-1 also exhibits an anti-migratory effect onneural progenitor cell (Mizuno et al. 2005) and endothelialcells (Castanares et al. 2007). These findings suggest that thebiological effects of ET-1 may be specific differences underdistinct conditions.

In conclusion, we demonstrated that ET-1 induces proM-MP-9 expression via activation of ETB receptors coupled toGi/o and Gq and transcription factors like Elk-1, NF-jB, andAP-1 (c-Jun/c-Fos), resulting in the promotion of cellmigration in astrocytes. Based on the observations fromliteratures and our findings, Fig. 8 depicts a model for themolecular mechanisms implicated in ET-1-induced proM-MP-9 expression in RBA-1 cells. These findings imply thatET-1 functionally regulates the progression of brain diseases.The molecular mechanisms underlying ET-1-induced proM-MP-9 expression and consequent cell migration may providepotential targets for the treatment of brain injury.

Acknowledgements

The authors appreciated Dr. M. Karin (Department of Pharmacol-

ogy, University of California at San Diego, CA) and Dr. C.P.

Tseng (Chang Gung University, Taiwan) for providing dominant

negative mutant of IKKa/b and shRNAs (p42, c-Jun, and c-Fos),respectively. This work was supported by National Science

Council, Taiwan; Grant numbers: NSC98-2321-B-182-004 (CMY)

and NSC98-2320-B-255-001-MY3 (HLH) and Chang Gung

Medical Research Foundation; Grant numbers: CMRPD150313,

CMRPD140252, CMRPD170491, and CMRPD180371, CMRPD

150253 (CMY), and CMRPF170022 (HLH).

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department of pharmacology, chang gung university, tao ... · and blood-brain barrier (bbb)...

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